Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation, dependence on extracellular and intracellular Na+, kinetics, pH dependency and the effect of cell volume and N-ethylmaleimide

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Abstract
Summary The effect of extracellular and intracellular Na+ (Na + o , Na + i ) on ouabain-resistant, furosemide-sensitive (FS) Rb+ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na++1 K++2 Cl−) cotransport system operates in two different modes: modei) promoting bidirectional 1∶1 (Na+−K+) cotransport, and modeii) a Na + o -independent 1∶1 K + o /K + i exchange requiring Na + i which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb+ uptake through Rb + o /K + i exchange contributes about 25% to total Rb+ uptake in 145mm NaCl media containing 5mm RbCl at normal Na + i (pH 7.4). Na+−K+ cotransport into the cells occurs largely additive to K+/K+ exchange. Inward Na+−Rb+ cotransport exhibits a substrate inhibition at high Rb + o . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K+/K+ exchange (apparent pK close to pH 7.4). The apparentK m Rb + o of Na+−K+ cotransport is low (2mm) and almost independent of pH, and high for K+/K+ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na++1 K++2 Cl−) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl−-dependent K+ transport pathway that is independent of both Na + o and Na + i . This pathway differs in many properties from the basal, Na + o -independent K+/K+ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na + o -independent FS K+ transport pathway which most probably is not identical to basal K+/K+ exchange. K + o + o + o 2+ o reduce furosemide-resistant Rb+ inward leakage relative to choline + o .